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(Circulation Research. 1996;79:14-19.)
© 1996 American Heart Association, Inc.

Articles

Cell Cycle-Dependent Expression of L- and T-Type Ca²⁺ Currents in Rat Aortic Smooth Muscle Cells in Primary Culture

Takeshi Kuga, Sei Kobayashi, Yoji Hirakawa, Hideo Kanaide, Akira Takeshita

the Research Institute of Angiocardiology and Cardiovascular Clinic (T.K., Y.H., A.T.) and the Division of Molecular Cardiology (S.K., H.K.), Faculty of Medicine, Kyushu University, Fukuoka, Japan.

Correspondence to Takeshi Kuga, MD, Research Fellow of Japanese Scientific Society, Research Institute of Angiocardiology and Cardiovascular Clinic, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812, Japan.

	Abstract

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The expression of L- and T-type Ca²⁺ channels has been reported to change during various biological events, including cellular differentiation and proliferation. The present study aimed to examine whether or not the expression of L- and T-type Ca²⁺ channels depends on the cell cycle in rat aortic smooth muscle cells in primary culture. Both the phase of the cell cycle and the functional expression of Ca²⁺ channels were determined in the same single cell, using an immunocytochemical analysis of cell cycle-specific nuclear antigens and a whole-cell voltage-clamp method, respectively. In the G₀ (n=130) and M (n=75) phases, all cells showed only L-type Ca²⁺ currents. The cells showing a T-type Ca²⁺ current appeared in the G₁ phase (37%, n=85) and increased in the S phase (90%, n=21). For L-type Ca²⁺ channels, the current density was significantly greater in the G₁ phase than in the G₀ and M phases. However, either the voltage-dependent properties or the dose-response relationships of Bay K 8644- and second messenger-induced modulations of L-type Ca²⁺ current did not differ in the four phases of the cell cycle. These findings thus indicate that the expression of L- and T-type Ca²⁺ channels depends on the cell cycle, whereas the characteristics of L-type Ca²⁺ channels do not differ between the phases of the cell cycle.

Key Words: Ca²⁺ channel • ion channel • proliferation • smooth muscle

	Introduction

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Two types (L- and T-type) of Ca²⁺ channels have been previously identified in various preparations. Expression of the types of Ca²⁺ channels changes with cellular differentiation, proliferation, or hypertrophy.^{1 2 3 4 5 6 7 8 9 10 11} In neurons,^{1 2 3} cardiac myocytes,^{4 5} and skeletal muscle cells,^{6 7} T-type Ca²⁺ channels are predominantly expressed at the early embryonic or neonatal stage; thereafter, they disappear with maturation. Moreover, T-type Ca²⁺ channels are reexpressed in adult myocytes when they are stimulated to an active growth phase by pressure overload⁸ or growth hormone.⁹ Rat aortic smooth muscle cells in primary culture show heterogeneous expression of L- and T-type Ca²⁺ channels: some cells express only one type of Ca²⁺ channel, whereas others express both L- and T-type Ca²⁺ channels.^{10 11 12 13 14} Some of those studies have also suggested that cells in the proliferative phases frequently express T-type Ca²⁺ channels.^{10 11} In contrast, most cells in the nonproliferative phases express only L-type Ca²⁺ channels. These findings support the hypothesis that expression of L- and T-type Ca²⁺ channels may differ between the phases of the cell cycle. Recent studies have suggested that expression of Cl^- channels^{15 16} and K⁺ channels^{17 18} differs depending on the cell-cycle phase, but the cell cycle-dependent difference in expression of Ca²⁺ channels has not been previously demonstrated.

The present study thus examined whether expression of L- and T-type Ca²⁺ channels differs between the phases of the cell cycle of rat aortic smooth muscle cells in primary culture. We recently developed a method to determine the phase of the cell cycle in an individual vascular smooth muscle cell.¹⁹ In the present study, we determined the phase of the cell cycle and the type of Ca²⁺ channels expressed in the same single cell. We also examined whether the characteristics of L-type Ca²⁺ channels differed between the phases of the cell cycle.

	Materials and Methods

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Cell Culture
Smooth muscle cells of the aortic media in male Wistar rats (250 to 300 g) were dispersed enzymatically and cultured on a chamber slide (Lab-Tek), as previously described.¹⁹ It was confirmed by electron microscopic examination and direct immunofluorescence stainings of smooth muscle myosin and actin that the cells were not contaminated by fibroblasts or endothelial cells.

Electrical Measurement
The experimental conditions under which current recordings were made were similar to those reported in our previous studies.^{10 12 13 14} Briefly, the cultured cells on a plate (No. 4804, Lab-Tek) were trypsinized for 1 minute and then were placed in a recording chamber. We performed the whole-cell voltage-clamp method using heat-polished glass patch electrodes (VC-H075P, Termo) with a tip resistance of 2 to 5 M to evaluate the functional expression of L- and T-type Ca²⁺ channels. The currents were amplified using a patch-clamp amplifier (EPC-7, List-Electronic) with capacitance and series-resistance compensation and filtered at 2.0 kHz. All experiments were monitored on a digital storage scope (DS-6121A, Iwatsu) and stored on a PCM data recorder (RP-880, Sony) for later analysis. The amplitudes of I_Ca were measured at the peak of each current. The amplitudes of I_Ca were estimated by subtracting leakage currents from recording currents. Leakage current was calculated with the current elicited by a small hyperpolarizing pulse.

We estimated the peak amplitude of the T-type I_Ca by step depolarization to -30 mV from a V_H of -100 mV. This step depolarization induced little or no activation of L-type Ca²⁺ channels.

When we estimated the peak amplitude of L-type I_Ca, we applied a step depolarization to 20 mV from a V_H of -60 mV. This step depolarization induced little or no activation of T-type I_Ca. Preliminary studies using Cd²⁺ (0.1 mmol/L) or F^- (5 mmol/L), which abolished L-type I_Ca but rarely affected T-type I_Ca,¹⁰ confirmed that the two protocols of step depolarizations were appropriate for separating the two types of I_Ca in rat aortic smooth muscle cells in primary culture. We could thus estimate the peak amplitude of both types of I_Ca separately by using the two-step depolarization protocols.^{10 12}

The steady state inactivation of L-type Ca²⁺ channels was obtained using a double-pulse protocol: from a V_H of -80 mV, the membrane was depolarized to different potential levels by a prepulse (3 seconds) before the constant test pulse (300 milliseconds) to 20 mV. The steady state activation of L-type Ca²⁺ channels was obtained by extrapolating the linear part of the corresponding I-V curve. The steady state inactivation and activation curves were deduced from the Boltzmann equation.

The internal solution contained (mmol/L) N-methyl-D-glucamine 110, Na₂ATP 5, MgSO₄ 5, TEA-Cl 20, HEPES 5, EGTA 10, and Tris base 2. The pH was adjusted to 7.2 by HCl. The external solution contained (mmol/L) CaCl₂ 20, NaCl 110, KCl 2, glucose 10, TEA-Cl 15, 4-aminopyridine 5, and HEPES 5. The pH was adjusted to 7.4 with Tris base. During the experiments, the chamber (0.5 mL) was continuously perfused with external solution at a rate of 1 mL/min. When we examined the effects of drugs such as Bay K 8644, the external solution containing drugs was perfused at a rate of 10 mL/min. The exchange of the external solution was accomplished within 30 seconds. All experiments were carried out at room temperature (20°C to 24°C).

Immunocytochemical Analysis of the Cell Cycle
After recording I_Ca, we removed the electrode from the cell and put a mark on the slide (Lab-Tek) around the cell with a needle. Then the cell-cycle phase of the cell enclosed by the mark was determined as described previously.¹⁹ Briefly, the cell on the Lab-Tek slide was rapidly fixed in -20°C ethanol (75%) for 30 minutes and was subsequently washed with and incubated in phosphate-buffered solution containing 1 mg/mL bovine serum albumin for 10 minutes at 25°C. Double-labeled immunofluorescence staining was carried out by incubation with monoclonal antibodies against cell cycle-specific nuclear antigens (fluorescein isothiocyanate-conjugated anti-PCNA and R-phycoerythrin-conjugated Ki-67) for 30 minutes at 25°C. We observed the cell with double staining under a fluorescence microscope (Axioskop, Zeiss) and photographed it for later analysis. Because the staining properties of PCNA (no expression in the G₀ phase, moderate expression in the G₁ or G₂ phase, and maximal expression in the S phase) and Ki-67 (no expression in the G₀ phase, weak and aggregated expression in the G₁ phase, increasing expression in the S phase, and maximal expression in the G₂ and M phases) were different among the cell-cycle phases; the color pattern of the nucleus produced by double staining differed.²⁰ The cells in the G₀ phase had no specific nuclear fluorescence, whereas the cells in the G₁ phase showed green fluorescence (PCNA) associated with aggregated spotty red-orange fluorescence (Ki-67). The cells in the M phase had red (Ki-67) fluorescence, and the cells in the S phase emitted both red (Ki-67) and green (PCNA) fluorescence, thus giving them a yellow appearance. Differentiated nuclear stainings were confirmed in vascular smooth muscle cells, which were arrested in the S or M phase by aphidicolin or TN-16, respectively.

In the present study, we determined the functional expression of Ca²⁺ channels using the whole-cell voltage-clamp method on 646 cells. In 338 of these 646 cells, we tried to determine the cell-cycle phase by an immunocytochemical analysis, and the cell-cycle phase could be determined in 311 of 338 cells. In the remaining 27 cells, the cell-cycle phase could not be determined because the cells were lost from the Lab-Tek slide during the fixation and staining procedures.

Materials
PDBu, dibutyryl cGMP, and dibutyryl cAMP were purchased from Sigma Chemical Co. Bay K 8644 and nifedipine were purchased from Wako Jun-yaku Kogyo. All drugs were prepared as a stock solution and were made to a final concentration with the external solution. Bay K 8644 and PDBu were dissolved in DMSO to make a stock solution. At the concentrations used (<0.1%), DMSO had no effect on L-type I_Ca. The monoclonal antibodies (fluorescein isothiocyanate-conjugated anti-PCNA and R-phycoerythrin-conjugated Ki-67) were purchased from Dako Japan.

Statistics
The data are expressed as mean±SEM. The cell population concerning the expression of L- or T-type I_Ca was then compared among the different cell-cycle phases using the ² test. The current density of L- or T-type I_Ca of the cell was compared among the different cell-cycle phases by a one-way ANOVA with Bonferroni's test. V_0.5 and slope factors in steady state inactivation or activation were compared by one-way ANOVA. The dose-response relationships of Bay K 8644-induced, PDBu-induced, or the dibutyryl cGMP-induced changes in L-type I_Ca were compared by two-way ANOVA. Values of P<.05 were considered to be significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell Cycle-Dependent Expression of L- and T-Type I_Ca
Rat aortic smooth muscle cells in primary culture express two distinct types (L- and T-type) of Ca²⁺ channel that can be determined by their characteristics, including voltage dependences, current kinetics, ion selectivities, sensitivities to various organic and inorganic Ca²⁺ antagonists, and modulation by various drugs.^{10 12 13 14} Under our experimental conditions, T- and L-type I_Ca can only be identified by differences in their voltage dependences and current kinetics, as previously described.¹⁰ In brief, L-type I_Ca can be identified by the findings that step depolarization to >-30 mV evokes a long-lasting type I_Ca from a V_H of -60 mV and that I_Ca reaches the peak by step depolarization to 20 mV (Fig 1a, 1b, and 1d). In contrast, T-type I_Ca can be identified by the findings that transient I_Ca is evoked by depolarization to >-60 mV from a V_H of -100 mV and that I_Ca reaches the peak by depolarization to -30 mV (Fig 1b and 1c). Three types of the cells were identified on the basis of the expression of the Ca²⁺ channels: cells expressing only L-type I_Ca (Fig 1a and 1d), those expressing both L- and T-type I_Ca (Fig 1b), and those expressing only T-type I_Ca (Fig 1c). The immunocytochemical analysis showed that the cell in panel a was in the G₀ phase, that in panel b was in the G₁ phase, that in panel c was in the S phase, and that in panel d was in the M phase.

View larger version (27K): [in this window] [in a new window]   Figure 1. Representative recordings of L- and T-type ICa in each phase of the cell cycle. In the graphs on the left, the I-V relationships were obtained by step depolarizations (300 milliseconds) from a VH of -100 mV () and -60 mV (). In the middle, the current recordings were carried out in the same single cell shown in the graphs, with the use of two types of depolarization, from a VH of -100 mV to -30 mV (step A) and from a VH of -60 mV to 20 mV (step B). Step A and step B selectively produced the maximum T- and L-type ICa, respectively. On the right are fluorescence micrographs, in which the cell-cycle phase was determined by the immunocytochemical staining: G0 (a), G1 (b), S (c), and M (d) phases.

Fig 2a summarizes the population of the cells showing L- and T-type I_Ca in each cell cycle. The total number of cells examined was 311. All cells in the G₀ phase (n=130) showed only L-type I_Ca. In the G₁ phase (n=85), most cells (63%) showed only L-type I_Ca; however, the remaining (37%) showed T-type I_Ca. In the S phase (n=21), most cells showed T-type I_Ca (90%). In the M phase (n=75), however, most cells (96%) showed only L-type I_Ca, whereas the few remaining cells (4%) had no detectable I_Ca. Thus, the expression of two types of Ca²⁺ channel depended on the phase of the cell cycle (P<.01). The current density of T-type I_Ca was significantly greater in the S phase than the G₁ phase (P<.001) (Fig 2b). The current density of L-type Ca²⁺ channels was significantly greater in the G₁ phase than the G₀ and M phases (P<.005), but there was no significant difference in the current density of L-type Ca²⁺ channels among the G₀, M, and S phases (Fig 2c).

View larger version (28K): [in this window] [in a new window]   Figure 2. The expression of L- and T-type ICa in each phase of the cell cycle. a, Population (percentage) of cells showing only L-type ICa (L), both L- and T-type ICa (L+T), and only T-type ICa (T). In the M phase, a few cells showed no detectable inward current (N). The population significantly depended on the cell cycle (P<.01, analyzed by 2 test). b, The density of T-type ICa. The current density was significantly greater in the S phase than that in the G0, G1, or M phase (P<.001, analyzed by one-way ANOVA with Bonferroni's test). The current density in the G1 phase was significantly greater than that in the G0 or M phase (P<.001). c, The density of L-type ICa. The current density was significantly greater in the G1 phase than that in the G0 or M phase (P<.005, analyzed by one-way ANOVA with Bonferroni's test). There was no significant difference in the densities among the G0, M, and S phases. The numbers of cells in each cell cycle were 130 in the G0 phase, 85 in the G1 phase, 21 in the S phase, and 75 in the M phase.

Expression of L- and T-Type I_Ca and the Duration of Cell Culture
Previous studies have demonstrated that the expression of L- and T-type I_Ca changes with the duration of the cell culture.^{10 11} Fig 3 shows the relationship between the duration of the culture and the population (percentage) of the cells showing T-type I_Ca of a total of 646 cells. Within 6 hours (the "zero" culture day in Fig 3), all cells showed only L-type I_Ca (n=35). The immunocytochemical analysis of the cell cycle, which was performed in 25 of 35 cells, showed that all 25 cells were in the G₀ phase. From 6 to 24 hours (the 6-hour culture day in Fig 3), 20 cells showed only L-type I_Ca, and three cells showed both L- and T-type I_Ca. The immunocytochemical analysis of the cell cycle of those cells, which was performed in 21 of 23 cells, showed that the cells showing only L-type I_Ca were in the G₀ phase (n=18), whereas the other three cells showing both L- and T-type I_Ca were in the G₁ phase. From culture days 1 to 12, during which time cultured cells were proliferating, cells in the G₁, S, or M phase increased, and the cells showing T-type I_Ca increased (14% at day 1 and 40% at day 2 in Fig 3). During this period, all cells in the G₀ phase showed only L-type I_Ca (n=56). After >12 days, the cultured cells generally reached confluence, and all cells showed only L-type I_Ca (n=48). The immunocytochemical analysis of the cell cycle, which was performed in 32 of 48 cells, showed that all cells were in the G₀ phase.

View larger version (16K): [in this window] [in a new window]   Figure 3. Expression of T-type ICa and the duration of cell culture. The horizontal axis indicates the duration of cell culture. Day 0 indicates a duration of <6 hours. The numbers in the figure indicate the numbers of cells examined.

Characteristics of L-Type I_Ca in the G₀, G₁, and M Phases
The characteristics of L-type I_Ca were compared among the different phases of the cell cycle. We first examined the voltage-dependent properties of L-type I_Ca. Among the G₀, G₁, and M phases, there were no significant differences in the current-voltage relationship (Fig 4a), the steady state inactivation (Fig 4b), or the steady state activation (Fig 4c) of L-type I_Ca.

View larger version (22K): [in this window] [in a new window]   Figure 4. Characteristics of L-type Ca2+ channels in each phase of the cell cycle. a, Normalized I-V relationships of L-type ICa in the G0 (, n=14), G1 (, n=5), and M (, n=7) phases from a VH of -60 mV. Normalized ICa was defined as the ratio of ICa to peak ICa. b, Steady state inactivation (finf) curves of L-type ICa in the G0 (, n=10), G1 (, n=10), and M (, n=7) phases. There were no significant differences in the inactivation parameters among the three phases (G0, V0.5=-31±2 mV and slope factor [k]=7.8±0.7; G1, V0.5=-34±3 mV and k=8.2±0.7; and M, V0.5=-32±2 mV and k=8.5±1.0). c, Steady state activation (dinf) curves of L-type ICa in the G0 (, n=10), G1 (, n=7), and M (, n=7) phases. There were no significant differences in the activation parameters among the three phases (G0, V0.5=-3.2±3.2 mV and k=7.0±0.5; G1, V0.5=-4.2±1.3 mV and k=7.2±0.2; and M, V0.5=-2.3±1.5 mV and k=7.4±0.6). d, e, and f, Concentration-response relationships of the Bay K 8644-induced (d) and PDBu-induced (e) potentiation of L-type ICa and those of the dibutyryl cGMP-induced inhibition (f) of L-type ICa in the G0 (), G1 (), and M () phases. There were no significant differences in these concentration-response relationships. The data are expressed as percent amplitude relative to the control values obtained before the application of the drug. Each point was obtained from four to nine cells. The total number of cells used for the Bay K 8644 study was 23 in the G0 phase, 18 in the G1 phase, and 21 in the M phase. The numbers of cells used for the PDBu study were 20, 15, and 18 in the G0, G1, and M phases, respectively. The numbers of cells for the cGMP study were 18, 15, and 19 in the G0, G1, and M phases, respectively. In these experiments, we used the cells showing only L-type ICa in each phase of the cell cycle.

Second, we examined the effect of Bay K 8644, a dihydropyridine Ca²⁺ channel agonist, on L-type I_Ca. Bay K 8644 concentration-dependently increased L-type I_Ca, and the maximum effects were achieved at a concentration of >1 µmol/L. The concentration-response relationships of the Bay K 8644-induced augmentation of L-type I_Ca were similar among the cell-cycle phases (Fig 4d). When L-type I_Ca was maximally augmented by Bay K 8644 (1 to 10 µmol/L), the current density of L-type I_Ca was significantly greater in the G₁ phase (46.1±11.7 µA/cm²) than in the G₀ phase (14.9±3.5 µA/cm²) or the M phase (13.5±2.3 µA/cm²) (P<.05). We also evaluated the inhibitory effect of nifedipine, a dihydropyridine Ca²⁺-channel antagonist, on L-type I_Ca. No significant difference was observed in the concentration-response relationship of the nifedipine-induced inhibition of L-type I_Ca among the different phases of the cell cycle (data not shown).

Third, we examined the effect of second messengers on L-type I_Ca. PDBu, a protein kinase C-activating phorbol ester, increased L-type I_Ca in a concentration-dependent manner. No significant difference was found in the concentration-response relationship of the PDBu-induced augmentation between the phases of the cell cycle (Fig 4e). Dibutyryl cGMP, a membrane-permeable analogue of cGMP, inhibited L-type I_Ca in a concentration-dependent manner. No significant difference was observed in the concentration-response relationship of the cGMP-induced inhibition between the phases of the cell cycle (Fig 4f). In this preparation, dibutyryl cAMP (10 to 1000 µmol/L), a membrane-permeable analogue of cAMP, had no effects on L-type I_Ca in any phase of the cell cycle (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrated for the first time the cell cycle-dependent expression of L- and T-type I_Ca using vascular smooth muscle cells in primary culture. The characteristics of L-type I_Ca did not differ between the phases of the cell cycle. The selective expression of T-type I_Ca in the G₁ and S phases, but not in the G₀ phase, is also consistent with the results of the previous studies demonstrating the increased expression of T-type I_Ca in the proliferating cells.^{1 2 3 4 5 6 7 8 9} The cell cycle-dependent expression of T- and L-type I_Ca can also explain the finding that the expression of the two types of I_Ca changes with the duration of culture in this preparation.^{10 11} All cells within 6 hours of culture, which were considered to be freshly dispersed cells, were in the G₀ phase and showed only L-type I_Ca. Therefore, smooth muscle cells in the rat aorta in vivo may be in the G₀ phase and have only L-type Ca²⁺ channels. Moreover, all cells in the G₀ phase showed only L-type I_Ca regardless of the difference in the duration of culture, thus indicating that the expression of L- and T-type I_Ca does not depend on the duration or the state of cell culture, but instead depends on the cell cycle.

The difference in the density of L-type I_Ca could be the result of either the change in the open probability or the change in the number of the channels. We cannot clarify which mechanism might be responsible, but it appears unlikely that the difference in the current density resulted from the change in the open probability. First, we examined the density of L-type I_Ca after the maximum augmentation of the open probability of L-type Ca²⁺ channels by Bay K 8644.²¹ Even after the maximum augmentation of open probability, the density of L-type I_Ca was greater in the G₁ phase than in the G₀ or M phase. Second, we examined the density of L-type I_Ca after modification of the open probability by protein kinases. It has been shown that the open probability of L-type Ca²⁺ channels may be modified by protein kinases.^{14 22} Protein kinase C¹⁴ increases and protein kinase G²² decreases the open probability of L-type Ca²⁺ channels. It also has been shown that the activity of protein kinases changes with the cell-cycle progression.²³ However, the effects of PDBu (an activator of protein kinase C) or dibutyryl cGMP on L-type I_Ca did not differ among the phases of the cell cycle. It is known that cAMP potentiates L-type I_Ca in cardiac myocytes. In smooth muscle cells, however, the findings on the effect of cAMP on L-type I_Ca are not consistent. Potentiation,²⁴ inhibition,²⁵ dual effects (potentiation and inhibition),²² and the absence of any effect²⁶ have been reported. The tissue diversity and the difference in experimental conditions may account for the reported differences in the modulation of L-type I_Ca by cAMP in smooth muscle cells. In our preparation, dibutyryl cAMP (up to 1 mmol/L) had no significant effect on L-type I_Ca at any phase of the cell cycle. These findings suggest that the modification of the open probability of L-type I_Ca by protein kinases cannot explain the cell cycle-dependent difference in the density of L-type I_Ca. Therefore, we consider the possibility that the cell cycle-dependent changes in the density of L-type I_Ca may be due to the changes in the channel number, as previously suggested in the cell cycle-dependent changes in other ion channels.^{15 17 18}

The major implications of the present study are as follows: First, the cell cycle-dependent expression of L-type Ca²⁺ channels may influence the smooth muscle functions related to Ca²⁺ influx, such as contraction, because the L-type Ca²⁺ channels play a major role in the Ca²⁺ influx in smooth muscle cells. Second, the predominant expression of T-type Ca²⁺ channels in the G₁ and S phases but not in the G₀ phase suggests that T-type Ca²⁺ channels may play a role in the cellular proliferation, although the precise role of T-type Ca²⁺ channels in smooth muscle cells remains unknown at present. Further studies are needed to clarify the precise role of T-type Ca²⁺ channels. Third, the cell cycle-dependent expression of Ca²⁺ channels must be taken into consideration when the cellular characteristics relating Ca²⁺ channels in the cultured or proliferating cells are investigated.

In summary, this is the first study that has directly demonstrated the cell cycle-dependent expression of Ca²⁺ channels. Our findings are potentially important in the understanding of the pathophysiology of various disease states, such as atherosclerosis, restenosis after angioplasty, and vascular spasm, because the proliferation of vascular smooth muscle cells plays an important role in those diseases and because the Ca²⁺ channels serve diverse biological functions.

	Selected Abbreviations and Acronyms

 

DMSO = dimethyl sulfoxide I-V = current (I)-voltage (V) ICa = Ca2+ current PCNA = proliferating cell nuclear antigen PDBu = phorbol-12,13-dibutyrate TEA = tetraethylammonium V0.5 = half-maximal activation (inactivation) VH = holding potential

	Acknowledgments

 
This study was supported in part by a grant from the Japan Society for the Promotion of Science for Japanese Junior Scientists; by a grant from the Kaibara Morikazu Medical Science Promotion Foundation; by a grant from the Kanae Foundation of Research for New Medicine; by Kimura Memorial Heart Foundation Research Grant for 1995; and by grants-in-aid for Developmental Scientific Research (No. 06557045), for General Scientific Research (Nos. 07407022 and 07833008), for Scientific Research on Priority Areas (Nos. 06274221 and 06262219), and for Creative Basic Research "Studies of Intracellular Signaling Network" from the Ministry of Education, Science and Culture, Japan. We thank Brian Quinn for reading the manuscript.

Received December 15, 1995; accepted April 8, 1996.

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